Weyman C M, Ramocki M B, Taparowsky E J, Wolfman A
Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
Oncogene. 1997 Feb 13;14(6):697-704. doi: 10.1038/sj.onc.1200874.
Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30-40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.
在23A2骨骼肌成肌细胞中致癌性Ras的表达足以诱导转化表型和分化缺陷表型，但致癌性Ras在这些细胞中激活的信号通路及其对每种表型的各自贡献尚未得到探索。在本研究中，我们研究了对照23A2成肌细胞以及通过稳定表达致癌性（G12V）Ha-ras基因（Ras9细胞）而导致分化缺陷的23A2成肌细胞中的丝裂原活化蛋白激酶（MAP激酶）活性。从Ras9细胞中免疫沉淀的MAP激酶比对照23A2细胞中的活性高30 - 40%。为了确定这种升高的MAP激酶活性对于维持致癌性Ras诱导的表型是否必不可少，我们使用了选择性MAP激酶激酶1（MEK1）抑制剂PD 098059。PD 098059将Ras9细胞中的MAP激酶活性降低到23A2细胞中的水平。PD 098059不影响23A2成肌细胞的分化能力。PD 098059使Ras9细胞的转化形态恢复正常，但未恢复这些细胞表达肌肉特异性肌球蛋白重链基因或形成肌纤维的能力。用PD 098059处理也不影响致癌性Ha-Ras在共表达MyoD的C3H10T1/2细胞中建立非肌源性表型的能力。这些结果表明，MAP激酶的激活对于Ras9细胞的转化形态是必需的，但致癌性Ras建立或维持分化缺陷表型并不需要MAP激酶的激活。虽然这些数据不排除MEK1或MAP激酶的组成性信号传导可能抑制成肌细胞分化的可能性，但它们清楚地表明，致癌性Ras激活的其他途径足以抑制分化。
